Because of the high incidence of adverse reactions to standard anti-Pneumocystis carinii chemotherapeutic agents, and the failure of such agents in 10-30% of AIDS patients with Pneumocystis carinii pneumonia, the development of effective alternative therapeutic agents is needed. Standard drug screening has relied on the use of an animal model, which is expensive and cumbersome, and thus in vitro cultures systems are needed. Based on earlier studies, we have developed an in vitro culture model that relies on metabolism by Pneumocystis carinii of paraminobenzoic acid (PABA). Sensitivity to a drug is determined by including the drug in this culture system and evaluating the amount of inhibition of radiolabelled PABA incorporation into reduced Folates compared to controls. Using this system, we have screened a number of inhibitors of 2 enzymes, dihydrofolate reductase and dihydropteroate synthetase, and continue to screen such agents as well as additional agents such as pentamidine analogues. One drug identified as a potential inhibitor of DHPS is para- acetamidobenzoic acid, an analogue of PABA. Animal studies with this drug are currently underway. We are also currently evaluating an alternative in vitro culture system that depends on incorporation of radiolabeled methionine into P. carinii proteins. Such an assay system will allow a more rapid screening of a larger number of chemotherapeutic agents, and at the same time will reduce the number of animals that need to be utilized in drug screening studies. The ultimate goal is to develop drugs that are more effective or less toxic than those currently available for treating Pneumocystis carinii pneumonia.